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Updated: Apr 27, 2026
Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation
Published on: March 2, 2016
Zyxin modulates the transmigration of Haemophilus influenzae to the central nervous system
Yuko Miyazaki1, Takashi Yusa2, Saburo Matsuo3
1Department of Endocrinology and Metabolism; Yokohama City University School of Medicine; Yokohama, Kanagawa, Japan.
Abstract:
The mechanism by which Haemophilus influenzae causes meningitis is unclear. Previously, we established murine meningitis by intranasal instillation of H. influenzae as a cell-bound organism (CBO). In this study, we aimed to identify the molecules associated with inhibiting the transmigration of cells across the blood-brain barrier (BBB). Two-dimensional difference gel electrophoresis and protein identification by mass spectrometry were used for proteomic analysis. Analysis of the membranous extract from a tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cell (HBMEC) monolayer revealed 41 differentially expressed proteins. Zyxin, which is thought to be essential for tight cell-to-cell junctions, decreased 1.8-fold in TNF-α-treated HBMECs. In addition, zyxin transcript levels decreased 1.5-fold in cells derived from TNF-α-treated HBMECs. Intranasal instillation of CBOs in zyxin-deficient mice resulted in a significant higher mortality rate than in wild-type mice. Transmigration of CBOs across a HBMEC monolayer pretreated with TNF-α (1 ng/mL), interleukin (IL)-1β (10 ng/mL), or lipopolysaccharide (LPS; 10 ng/mL) was assayed by counting CBOs that migrated from an upper chamber into a lower chamber. HBMEC pretreated with TNF-α exhibited significantly greater migration (P<0.01) than did control cells or cells treated with IL-1β or LPS. Our findings highlight that zyxin is an important protein protecting the tight junction of the BBB against cell transmigration across the BBB. Finally, TNF-α produced in respiratory infection when the primary infection reached the BBB caused decreased zyxin levels in BBB cell membranes. Furthermore, H. influenzae reaching the BBB as CBOs could transmigrate into cerebrospinal fluid across the zyxin-decreased BBB.
Insights
Zyxin protein levels decrease during Haemophilus influenzae meningitis, weakening the blood-brain barrier (BBB). This allows bacteria to cross into the cerebrospinal fluid, increasing mortality in zyxin-deficient mice.
Area of Science:
- Neuroscience
- Infectious Diseases
- Cell Biology
Background:
- The precise mechanisms of Haemophilus influenzae meningitis remain incompletely understood.
- Previous research established a murine meningitis model using intranasal instillation of cell-bound organisms (CBOs).
Purpose of the Study:
- To identify molecules that inhibit cellular transmigration across the blood-brain barrier (BBB).
- To investigate the role of zyxin in BBB integrity during H. influenzae infection.
Main Methods:
- Proteomic analysis using 2D gel electrophoresis and mass spectrometry on tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cells (HBMECs).
- Assessing CBO transmigration across HBMEC monolayers pretreated with TNF-α, IL-1β, or LPS.
- Evaluating mortality rates in zyxin-deficient and wild-type mice after intranasal CBO instillation.
Main Results:
- Proteomic analysis revealed 41 differentially expressed proteins in TNF-α-treated HBMECs, with zyxin showing a significant decrease (1.8-fold).
- Zyxin transcript levels also decreased (1.5-fold) in TNF-α-treated HBMECs.
- HBMECs pretreated with TNF-α showed significantly greater CBO migration compared to controls or cells treated with IL-1β or LPS.
- Zyxin-deficient mice exhibited a higher mortality rate when infected with CBOs.
Conclusions:
- Zyxin is crucial for maintaining the integrity of tight junctions in the BBB, preventing cellular transmigration.
- TNF-α, produced during respiratory infections that reach the BBB, reduces zyxin levels in BBB cells.
- Reduced zyxin levels facilitate H. influenzae CBO transmigration across the BBB into the cerebrospinal fluid, contributing to meningitis and increased mortality.
